Ca2+ controls numerous cellular processes in skeletal muscle and alterations in Ca2+ homeostasis are associated with human diseases such as Duchenne Muscular Dystrophy (DMD), Malignant Hyperthermia (MH) and Central Core Disease (CCD). Defining the molecular mechanisms regulating intracellular Ca2+ signaling is a crucial step for developing new therapeutic interventions in these myopathies. The release of Ca2+ from sarcoplasmic reticulum (SR) via Ca2+ release channels (ryanodine receptors, RyRs) is a key step in skeletal muscle excitation-contraction coupling (ECC). It is triggered through a direct interaction of the plasmalemmal voltage sensors with RyRs and it is thought to be amplified by Ca2+-induced Ca2+ release (CICR), manifest as Ca2+ sparks. However, mature mammalian muscle does not display Ca2+ sparks during physiological ECC but it develops spontaneous spark activity under various pathophysiological conditions. The molecular events that lead to Ca2+ spark generation in mammalian muscle are unknown. Understanding these mechanisms is a prerequisite to prevent changes in Ca2+ homeostasis associated with a number of human muscle diseases. Our data suggest that reactive oxygen and nitrogen species (ROS/RNS) and mitochondria are key regulators of intracellular Ca2+ signaling in skeletal muscle. They have led us to the following hypotheses: 1). Under physiological conditions, the appearance of sparks is suppressed by reduced cytosolic environment, which maintains a low activity of RyR1, and by mitochondrial Ca2+ uptake. 2). Increased cytosolic Ca2+ levels promote ROS/RNS production through mitochondrial Ca2+ overload and/or stimulation of ROS/RNS production by other cellular sources. 3). ROS/RNS stimulate spark production by enhancing the Ca2+ release activity of RyR1 and/or by inhibiting mitochondrial Ca2+ uptake. 4). Cytosolic Ca2+ levels are elevated in MH due to SR Ca2+ leak, and in DMD due to increased Ca2+ influx. In both disorders, the outcome of increased cytosolic Ca2+ is: a) enhanced ROS/RNS production b) oxidative modification of RyR1, c) enhanced Ca2+ sensitivity of the modified RyR1 and d) the appearance of Ca2+ sparks. To test these hypotheses, we will carry out the following Specific Aims using electrophysiological methods and state-of-the-art imaging techniques (single and two-photon confocal imaging, digital photometry, UV-laser flash photolysis of caged compounds). We propose to: 1). Determine the mechanisms connecting cytosolic Ca2+ signals, mitochondrial Ca2+ uptake and ROS/RNS generation in muscle under physiological conditions. 2). Define how altered ROS/RNS generation affect cellular Ca2+ homeostasis in muscle from MH-susceptible and mdx mice (a mice model of DMD). PUBLIC HEALTH RELEVANCE: Release of Ca2+ from intracellular Ca2+ stores is a key step of excitation-contraction coupling. Alterations in skeletal muscle excitation-contraction coupling are associated with human diseases such as Hypokalemic Periodic Paralysis, Malignant Hyperthermia and Central Core Disease and, hence, defining the molecular mechanisms regulating the primary event in ECC is crucial for developing new therapeutic interventions in these diseases. The proposed experiments will provide us with new information about the mitochondrial control of Ca2+ signaling in skeletal muscle, and bring us one step closer to the understanding of a whole range of hereditary and acquired muscle disorders associated with metabolic and mitochondrial dysfunctions and Ca2+ mishandling.